Method of making a mycoplasma vaccine

ABSTRACT

The present invention relates to a method for the preparation of an immunogenic composition for the treatment and/or prophylaxis of  mycoplasma  infections in a subject comprising the cultivation of  mycoplasma  bacteria in a serum-reduced or swine serum-free, eukaryotic cell system; obtaining an antigen of the  mycoplasma  bacteria; and addition of a pharmaceutically acceptable carrier. Further, the present invention relates to the immunogenic composition obtainable by said method and a method for immunizing a subject comprising the administration of said immunogenic composition to a subject.

BACKGROUND

Bacteria of the Mycoplasma genus belong to the class Mollicutes andrepresent a group of organisms that derived from the Firmicutes lineage.Mollicutes are the smallest autonomously replicating organisms, whichdiffer structurally from other eubacteria in that they lack a cell wall.The surface of their single membrane is considered a key interface inmediating adaptation and survival in the context of a complex,immunocompetent host. Further, Mollicutes have a small genome and alimited number of metabolic pathways. Therefore, members of theMycoplasma genus have also been portrayed as “minimal self-replicatingorganisms.” However, despite this apparent simplicity, a large number ofmycoplasma bacteria are pathogens of humans and a wide range of animals.In contrast to other pathogenic bacteria where virulence is mostlydetermined by toxins, invasins, and cytolysins, pathogenic Mycoplasmabacteria appear to have no such typical primary virulence factors(Chambaud, I. et al, 2001, Nucleic Acids Res. 29: 2145-2153, Fraser etal, 1995, Science 270: 397-403). There is currently little knowledgeavailable on the molecular mechanisms and the effectors that allowpathogenic mycoplasmas to cause host cell damage, inflammation anddisease.

Pathogenic Mycoplasma bacteria cause mainly atypical pneumonia,uro-genital infections and arthritis in humans and in animals(Blanchard, A., and G. F. Browning (eds.). 2005. Mycoplasmas: Molecularbiology, pathogenicity and strategies for control. Horizon Bioscience,Wymondham U. K.; Kobisch M. and Friis N. F. 1996, Swine mycoplasmoses,Rev. Sci. Tech. Off. Int. Epiz. 15, 1569-1605). It is known thatreactivation or exacerbation of the signs repeats and transfersgradually to a chronic disease, and thus along with early diagnosis andearly treatment, prevention or treatment of exacerbation or reactivationare important. M. hyopneumoniae is the aetiological agent of enzooticpneumonia. In swine it is one of the most common and economicallyimportant diseases due to reduced weight gain and poor feed efficiency.The disease causes lesions in the lungs, a chronic cough, dull haircoat, retarded growth and unthrifty appearance lasting several weeks.The lung lesions, particularly in ventral apical and cardiac lobes, arecharacterized by a hyperplasia of the epithelial cells and an increasedperivascular and peribronchiolar accumulation of mononuclear cells. M.hyorhinis, another common mycoplasma of the respiratory tract of pigs,can cause polyserositis and arthritis in piglets. M. hyosynoviae isgenerally located in the tonsils and can cause arthritic disease,leading to economic losses. M. hyosynoviae is isolated from the jointsand pharyngeal/tonsillar samples and can induce antibodies in blood andjoint fluid. M. bovis is considered to be one of the more pathogenicbacteria of Mycoplasma and causes significant economic losses worldwide.Mycoplasma bacteria cause severe clinical signs in cattle of all ages.M. bovis is the most frequent Mycoplasma pathogen found to causepneumonia, mastitis, and arthritis in cattle and its etiological rolehas also been associated with otitis, keratoconjunctivitis, synovitis,and reproductive disorders in cows and bulls.

Because mycoplasma lack a cell wall, they are unaffected by many commonantibiotics such as penicillin or other beta-lactam antibiotics thattarget cell wall synthesis. Therapeutic agents for mycoplasma infectionthat are in practical use are some antibiotics such as macrolide-based,or new quinolone-based, or tetracycline-based antibiotics, but suchantibiotics have great adverse effects such as advent of drug-resistantstrains, which leads the mycoplasma infection to become severe whilesufficient treating effects are not expected, and becomes a cause fortransfer to a chronic disease. Further, vaccination is an effectivemethod of controlling mycoplasma infection. However, significant highyields of mycoplasma needed for the preparation of vaccines are obtainedby cultivation normally only in complex media (Kobisch M. and Friis N.F. 1996, Swine mycoplasmoses, Rev. Sci. Tech. Off. Int. Epiz. 15,1569-1605; Gabridge M. G. et al. 1976, Cultivation of mycoplasma in amodified tissue culture medium, Applied and Environmental microbiology.31, 986-989; Sotoodehnia A. Et al 2007, Preparation of agalactia vaccinein fermentor, Archives of rani institute, 62, 45-48; Dahlia et al., 2009Isolation of Mycoplasma hyosynoviae from pneumonic lung of swine,Tropical Biomedicine 26:341-345). Dependent on the mycoplasma bacteriato be cultivated, the complex media are supplemented with 10-30% serumand sometimes with yeast extract. Thus, due to the high price of serumthe cultivation of mycoplasma bacteria is cost intensive. Further, areduction of serum in the complex media would be beneficial in light ofanimal welfare, too. Thus, there is a need for the cultivation ofsignificant high yields of mycoplasma in a serum-reduced culturingsystem for the preparation of immunogenic compositions effective inpreventing mycoplasma infection. Moreover, the swine specific mycoplasmabacteria are generally cultivated in complex media containing swinespecific serum (Kobisch M. and Friis N. F. 1996, Swine mycoplasmoses,Rev. Sci. Tech. Off. Int. Epiz. 15, 1569-1605). However, swine specificserum can contain other swine-specific pathogens or can containantibodies against swine specific pathogens which can cause a reducedimmunogenicity of the immunogenic composition to be prepared. Thus,there is also a need for the cultivation of significant high yields ofmycoplasma in a swine serum-free culturing system for the preparation ofimmunogenic compositions effective in preventing mycoplasma infection.

DESCRIPTION OF THE INVENTION

Before the aspects of the present invention are described, it must benoted that as used herein and in the appended claims, the singular forms“a”, “an”, and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a or an antigen”includes a plurality of antigens, reference to the “cell” is a referenceto one or more cells and equivalents thereof known to those skilled inthe art, and so forth. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods, devices, and materials are nowdescribed. All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the cell lines,vectors, and methodologies as reported in the publications which mightbe used in connection with the invention. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

The present invention solves the problems inherent in the prior art andprovides a distinct advance in the state of the art.

Generally, the present invention provides a method for the preparationof an immunogenic composition for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation ofmycoplasma bacteria in a serum-reduced, eukaryotic cell system; b)obtaining an antigen of the mycoplasma bacteria; and c) addition of apharmaceutically acceptable carrier.

Advantageously, the experimental data provided by the present inventiondisclose that mycoplasma bacteria can be produced in a serum-reduced,eukaryotic cell system.

The term “immunogenic composition” refers to a composition thatcomprises at least one antigen, which elicits an immunological responsein the host to which the immunogenic composition is administered. Suchimmunological response can be a cellular and/or antibody-mediated immuneresponse to the immunogenic composition of the invention. The host isalso described as “subject”. Preferably, any of the hosts or subjectsdescribed or mentioned herein is an animal.

Usually, an “immunological response” includes but is not limited to oneor more of the following effects: the production or activation ofantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells and/or gamma-delta T cells, directed specifically toan antigen or antigens included in the immunogenic composition of theinvention. Preferably, the host will display either a protectiveimmunological response or a therapeutic response.

A “protective immunological response” will be demonstrated by either areduction or lack of clinical signs normally displayed by an infectedhost, a quicker recovery time and/or a lowered duration of infectivityor lowered pathogen titer in the tissues or body fluids or excretions ofthe infected host.

In case where the host displays a protective immunological response suchthat resistance to new infection will be enhanced and/or the clinicalseverity of the disease reduced, the immunogenic composition isdescribed as a “vaccine”.

An “antigen” as used herein refers to, but is not limited to, componentswhich elicit an immunological response in a host to an immunogeniccomposition or vaccine of interest comprising such antigen or animmunologically active component thereof. The antigen or immunologicallyactive component can be a microorganism that is whole (in inactivated ormodified live form), or any fragment or fraction thereof, which, ifadministered to a host, can elicit an immunological response in thehost. The antigen can be or can comprise complete live organisms ineither its original form or as attenuated organisms in a so calledmodified live vaccine (MLV). The antigen can further compriseappropriate elements of said organisms (subunit vaccines) whereby theseelements are generated either by destroying the whole organism or thegrowth cultures of such organisms and subsequent purification stepsyielding in the desired structure(s), or by synthetic processes inducedby an appropriate manipulation of a suitable system like, but notrestricted to bacteria, insects, mammalian or other species, andoptionally by subsequent isolation and purification procedures, or byinduction of said synthetic processes in the animal needing a vaccine bydirect incorporation of genetic material using suitable pharmaceuticalcompositions (polynucleotide vaccination). The antigen can comprisewhole organisms inactivated by appropriate methods in a so called killedvaccine (KV). If the organism is a bacterium, the killed vaccine iscalled a bacterin.

The term “treatment and/or prophylaxis” refers to the lessening of theincidence of the particular mycoplasma infection in a herd or thereduction in the severity of clinical signs caused by or associated withthe particular mycoplasma infection. Thus, the term “treatment and/orprophylaxis” also refers to the reduction of the number of animals in aherd that become infected with the particular mycoplasma bacteria(=lessening of the incidence of the particular mycoplasma infection) orto the reduction of the severity of clinical signs normally associatedwith or caused by a mycoplasma infection in a group of animals whichanimals have received an effective amount of the immunogenic compositionas provided herein in comparison to a group of animals which animalshave not received such immunogenic composition.

The “treatment and/or prophylaxis” generally involves the administrationof an effective amount of the immunogenic composition of the presentinvention to a subject or herd of subjects in need of or that couldbenefit from such a treatment/prophylaxis. The term “treatment” refersto the administration of the effective amount of the immunogeniccomposition once the subject or at least some animals of the herd is/arealready infected with such mycoplasma and wherein such animals alreadyshow some clinical signs caused by or associated with such mycoplasmainfection. The term “prophylaxis” refers to the administration of asubject prior to any infection of such subject with mycoplasma or atleast where such animal or none of the animals in a group of animals donot show any clinical signs caused by or associated with the infectionby such mycoplasma.

The term “an effective amount” as used herein means, but is not limitedto an amount of antigen, that elicits or is able to elicit an immuneresponse in a subject. Such effective amount is able to lessen theincidence of the particular mycoplasma infection in a herd or to reducethe severity of clinical signs of the particular mycoplasma infection.

Preferably, clinical signs are lessened in incidence or severity by atleast 10%, more preferably by at least 20%, still more preferably by atleast 30%, even more preferably by at least 40%, still more preferablyby at least 50%, even more preferably by at least 60%, still morepreferably by at least 70%, even more preferably by at least 80%, stillmore preferably by at least 90%, and most preferably by at least 95% incomparison to subjects that are either not treated or treated with animmunogenic composition that was available prior to the presentinvention but subsequently infected by the particular mycoplasmabacteria.

The term “clinical signs” as used herein refers to signs of infection ofa subject from mycoplasma bacteria. The clinical signs of infectiondepend on the pathogen selected. Examples for such clinical signsinclude but are not limited to respiratory distress, polyserositis (suchas peritonitis, pleuritis, pericarditis), arthritis (lameness andswollen joints), otitis, roughened hair coat, slight fever, depression,reduced appetite, and bacteremia. However, the clinical signs alsoinclude but are not limited to clinical signs that are directlyobservable from a live animal. Examples for clinical signs that aredirectly observable from a live animal include nasal and oculardischarge, lethargy, coughing, wheezing, thumping, elevated fever,weight gain or loss, dehydration, diarrhea, joint swelling, lameness,wasting, paleness of the skin, unthriftiness, diarrhea, and the like.

Reducing the incidence of or reducing the severity of clinical signscaused by or being associated with the particular mycoplasma infectionin a subject can be reached by the administration of one or more dosesof the immunogenic composition of the present invention to a subject inneed. As demonstrated by the Examples 2 and 3, the immunogeniccomposition as provided herein has been proven to be efficacious afterthe administration of a single dose to a subject of need.

The term “infection” or “infected” refer to the infection of a subjectby a pathogen, i.e. M. hyorhinis or M. hyorhinis and M. hyosynoviae orM. hyorhinis, M. hyopneumoniae and M. hyosynoviae.

The term “mycoplasma” is known by the person skilled in the art.“Mycoplasma” refers to a genus of bacteria, e.g. as described inBlanchard, A., and G. F. Browning (eds.). 2005. Mycoplasmas: Molecularbiology, pathogenicity and strategies for control. Horizon Bioscience,Wymondham U.K.; Kobisch M. and Friis N. F. 1996, Swine mycoplasmoses,Rev. Sci. Tech. Off. Int. Epiz. 15, 1569-1605. Bacteria can beclassified based on their biochemical and microbiological properties aswell as their morphology. These classification criteria are well knownin the art. In general the mycoplasma infection is associated with theclinical signs described elsewhere in this description.

The term “mycoplasma” as used herein refers to M. hyorhinis or M.hyorhinis and M. hyosynoviae or M. hyorhinis, M. hyopneumoniae and M.hyosynoviae. However, the term mycoplasma also encompasses M. bovis.Complete genome sequence of M. hyorhinis is exemplarily provided e.g. byLiu, W. et al., J. Bacteriol. 2010, vol. 192 (21), 5844-45 doi:10.1128/JB.00946-10. Epub 2010 Aug. 27 or by Calcutt M J. et al., 2012,J. Bacteriol. Vol. 194 (7), 1848 doi: 10.1128/JB.00033-12. Isolates ofM. hyosynoviae are exemplarily deposited at the American Tissue CultureCollection under accession numbers ATCC 25591 or ATCC 27095. Isolates ofMycoplasma hyopneumoniae are exemplarily deposited at the AmericanTissue Culture Collection under accession numbers ATCC 25095, ATCC25617, and ATCC 25934. The genomic DNA of Mycoplasma hyopneumoniaeJ-strain is deposited at the American Tissue Culture Collection underaccession numbers ATCC 25934D. Isolates of Mycoplasma bovis are wellknown to a person skilled in the art and some isolates are exemplarilydeposited at the American Tissue Culture Collection under accessionnumbers ATCC 25025, ATCC 25523, and ATCC 27368.

The term “cultivation” is known by the person skilled in the art. Theterm relates to the propagation of cells in culture outside theorganism. Particularly, the term “cultivation” relates to thepropagation of cells outside the organism in a cell system.

The term “cell system” is known by the person skilled in the art. Inparticular, the term “cell system” is an in vitro cell culture systemfor the cultivation of microorganism, such as e.g. mycoplasma bacteria.Such cell system comprises host cells and cell culture medium suitablefor the propagation of such cells outside of the organism. Inparticular, the host cells may or may not be susceptible for aninfection with the mycoplasma bacteria. Such host cells can be presentas live cells, in inactivated form or as cell fragments. Preferably,such host cells are eukaryotic cells of a eukaryotic cell system.

The term “eukaryotic cell system” comprises primary eukaryotic cells andeukaryotic cells derived from multicellular organisms such as plants oranimals. Furthermore, eukaryotic cell system encompasses eukaryoticsingle cell organisms (also referred to as microorganisms), e.g.bacteria or fungi including yeast. However, it is understood theeukaryotic cells are different from mycoplasma bacteria. Eukaryotic hostcells that can be used to practice the method as described hereininclude but are not limited to Madin-Darby Canine Kidney Epithelial(MDCK) cells (e.g. Madin-Darby Canine Kidney Epithelial Cells asdeposited with the American Tissue Culture Collection under accessionnumber ATCC CCL-34 or ATCC CRL-2285) or McCoy cells (e.g. as depositedwith the American Tissue Culture Collection under accession number ATCCCRL-1696).

The term “cell free cultivation system” as used herein refers tocultivation system that does not include any cells except of themycoplasma bacteria.

The term “serum reduced” refers to a reduced amount of serum that isadded for the cultivation of mycoplasma bacteria in the eukaryotic cellsystem compared to the amount of serum that is used for the cultivationof mycoplasma bacteria of the same species in a cell free cultivationsystem. The amount of serum for the cultivation of mycoplasma bacteriain the eukaryotic cell system compared to the amount of serum for thecultivation of mycoplasma bacteria in a cell free cultivation system isreduced by at least 10%, preferably by at least 20%, more preferably byat least 30%, even more preferably by at least 40%, even more preferablyby at least 50%, even more preferably by at least 60%, even morepreferably by at least 70%, even more preferably by at least 80%, evenmore preferably by at least 90%, even more preferably by at least 95%,even more preferably by at least 96%, even more preferably by at least97%, even more preferably by at least 98%, even more preferably by atleast 99%, most preferably by 100%. Thus, it has to be understood thataccording to the present invention the mycoplasma bacteria are mostpreferably cultivated in a eukaryotic cell system lacking any serum.

Preferred amounts of serum for the cultivation of mycoplasma bacteria inthe eukaryotic cell system include serum concentrations between about0-10% (v/v), more preferably from about 1-9% (v/v), still morepreferably from about 1-8% (v/v), even more preferably from about 1-7%(v/v), even more preferably from about 1-6% (v/v), and most preferablyabout 2-5% (v/v).

Preferred amounts of serum for the cultivation of mycoplasma bacteria inthe eukaryotic cell system comprising MDCK cells include serumconcentration between about 0-6% (v/v), more preferably from about 1-5%(v/v), still more preferably from about 2-4% (v/v), even more preferablyfrom about 2-3% (v/v), and most preferably about 2% (v/v).

Preferred amounts of serum for the cultivation of mycoplasma bacteria inthe eukaryotic cell system comprising McCoy cells include serumconcentration between about 0-10% (v/v), more preferably from about 1-9%(v/v), still more preferably from about 2-8% (v/v), even more preferablyfrom about 3-7% (v/v), even more preferably from about 4-6% (v/v), andmost preferably about 5% (v/v).

In accordance with one aspect of the present invention it is to beunderstood that the eukaryotic cells of the eukaryotic cell system areto be infected with mycoplasma bacteria. The infection of eukaryoticcells by mycoplasma bacteria and the conditions of the post incubationperiod are well known to the person skilled in the art. However,preferably, post transfection the cells are incubated over a period ofup to 21 days, more preferably from about two days to about fourteendays, more preferably from about two days to about eight days, stillmore preferably from about three to five days. Preferred incubationconditions include a temperature between about 32-42° C., morepreferably from about 34-40° C., still more preferably from about 35-39°C., even more preferably from about 36-38° C., and most preferably about37° C. Preferred incubation conditions also include a CO₂-concentrationbetween about 2% to 8%, more preferably from about 3% to 7% even morepreferably from about 4% to 6%, and most preferably about 5%.Preferably, the eukaryotic cells are observed following transfection forcharacteristic changes, such as cell density trends, the decrease inviability including cytopathic effects during the post-infection periodand the color change of the medium due to pH-changes.

The term “obtaining” comprises the harvest, isolation, purificationand/or formulation (e.g. finishing, inactivation and/or blending) of theantigen.

The term “harvest” refers to collecting or recovering the antigen of themycoplasma bacteria from the transfected eukaryotic cell system. Anyconventional method known in the art can be used to recover saidmycoplasma antigen, e.g. any separation method. Well known methods inthe art comprise centrifugation or filtration, such as using asemi-permeable membrane having a certain pore size.

The term “isolation” comprises an isolation step of the mycoplasmaantigen. Methods for the isolation of antigens of the mycoplasmabacteria from the infected eukaryotic cell system are known to a personskilled in the art. Those methods comprise physical and/or chemicalmethods, including but are not limited to freeze thaw cycles, treatmentwith ultrasound and the alike.

Methods for the “purification” of antigens from the isolate are known toa person skilled in the art, for example by those methods described inProtein purification methods—a practical approach (E. L. V. Harris andS. Angel, eds., IRL Press at Oxford University Press). Those methodsinclude, but are not limited to, separation by centrifugation and/orfiltration, precipitation, size exclusion (gel filtration)chromatography, affinity chromatography, metal chelate chromatography,ion-exchange chromatography covalent chromatography, hydrophobicinteraction chromatography, and the alike. The antigen can be obtainedin a purified pure form, or free or substantially free of other cellularmaterials or culture medium etc. After said isolation and/orpurification the antigen exhibits a purity of at least 80%, preferably80%-90%, more preferably 90%-97%, most preferred more than 97% up to anabsolute pure form without any contamination.

According to a further aspect, “obtaining” as used herein can alsoinclude further finishing steps as part of the final formulationprocess, like the addition of buffer, inactivation, neutralization stepsand the alike.

Any conventional “inactivation” method can be used for purposes of thepresent invention. Thus, inactivation can be performed by chemicaland/or physical treatments which are known to the person skilled in theart. Preferred inactivation methods include the addition cyclized binaryethylenimine (BEI) including the addition of a solution of2-bromoethyleneamine hydrobromide (BEA), which has been cyclized tobinary ethylenimine (BEI). Preferred further chemical inactivationagents comprise but are not limited to Triton X-100, Sodiumdeoxycholate, Cetyltrimethylammonium bromide, β-Propiolactone,Thimerosal, Phenol and Formaldehyde (Formalin). However, theinactivation can also comprise a neutralization step. Preferredneutralization agents include but are not limited to sodium thiosulfate,sodium bisulfite and the alike.

Preferred formalin inactivation conditions include formalinconcentration from about 0.02% (v/v)-2.0% (v/v), more preferably fromabout 0.1% (v/v)-1.0% (v/v), still more preferably from about 0.15%(v/v)-0.8% (v/v), even more preferably from about 0.16% (v/v)-0.6%(v/v), and most preferably about 0.2% (v/v)-0.4% (v/v). Incubation timedepends on the resistance of the mycoplasma species. In general, theinaction process is performed until no mycoplasma growth can be detectedin a suitable cultivation system.

According to a further aspect, the inactivated bacterin component of theinvention can be incorporated into liposomes using known technology suchas that described in Nature, 1974, 252, 252-254 or Journal ofImmunology, 1978, 120, 1109-13. According to another aspect, theinactivated bacterin component of the invention can be conjugated tosuitable biological compounds such as polysaccharides, peptides,proteins, or the like, or a combination thereof.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced, eukaryotic cellsystem; b) obtaining an antigen of the mycoplasma bacteria; and c)addition of a pharmaceutically acceptable carrier, wherein theimmunogenic composition is a mycoplasma immunogenic composition.

In one aspect of the present invention the serum that is used for thecultivation of the mycoplasma bacteria is free of swine-serum. The term“free of swine-serum” means that no swine-serum is added during thecultivation process of the mycoplasma bacteria in the eukaryotic cellsystem.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced, eukaryotic cellsystem; b) obtaining an antigen of the mycoplasma bacteria; and c)addition of a pharmaceutically acceptable carrier, wherein the serum isfree of swine-serum. According to a further aspect, all of themycoplasma antigens are produced in a swine-serum free, eukaryotic cellsystem.

In a further aspect, the cultivation of the mycoplasma bacteria occursin the absence of serum. This means, that no serum is added during thecultivation of the mycoplasma bacteria in the eukaryotic cell system.Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a eukaryotic cell system in theabsence of serum; b) obtaining an antigen of the mycoplasma bacteria;and c) addition of a pharmaceutically acceptable carrier. According to afurther aspect, all of the mycoplasma antigens are produced in theabsence of serum.

In one aspect of the present invention the mycoplasma antigens is/are awhole inactivated mycoplasma bacterin. It is understood, that only one,some or all of the mycoplasma antigens can be whole inactivatedmycoplasma bacterin.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the mycoplasma antigen is a whole inactivated bacterin.According to a further aspect, all of the mycoplasma antigens within theimmunogenic composition are whole inactivated bacterins. The inactivatedmycoplasma antigens can be obtained in such that step b) of the methoddescribed hereinabove includes an inactivation step. Thus, according toa further aspect, method is provided for the preparation of animmunogenic composition for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation ofmycoplasma bacteria in a serum-reduced or swine serum free, eukaryoticcell system; b) obtaining an antigen of the mycoplasma bacteria andinactivating the mycoplasma antigens; and c) addition of apharmaceutically acceptable carrier.

The whole inactivated bacterin can be obtained by the inactivation ofwhole mycoplasma bacteria, preferably by a method as described hereinabove. Thus, according to one aspect, the present application provides amethod for the preparation of an immunogenic composition for thetreatment and/or prophylaxis of mycoplasma infections in a subjectcomprising a) cultivation of mycoplasma bacteria in a serum-reduced orswine serum free, eukaryotic cell system; b) obtaining an antigen of themycoplasma bacteria and inactivating the mycoplasma antigens; and c)addition of a pharmaceutically acceptable carrier, wherein themycoplasma antigens are complete mycoplasma bacteria.

Preferably, the mycoplasma bacteria are inactivated with formalin asdescribed hereinabove. Thus, according to one aspect, the presentapplication provides a method for the preparation of an immunogeniccomposition for the treatment and/or prophylaxis of mycoplasmainfections in a subject comprising a) cultivation of mycoplasma bacteriain a serum-reduced or swine serum free, eukaryotic cell system; b)obtaining an antigen of the mycoplasma bacteria and inactivating themycoplasma antigens with formalin; and c) addition of a pharmaceuticallyacceptable carrier, wherein the mycoplasma antigens are completemycoplasma bacteria. Preferably, the formalin is used in concentrationsas described hereinabove.

In one aspect of the present invention the eukaryotic cell systemcomprises a MDCK cell line. The MDCK (Madin-Darby Canine KidneyEpithelial Cells) cell line was derived from the kidney tissue of anadult female cocker spaniel. However, the MDCK cell line is known to theperson skilled in the art. Thus, according to one aspect, the presentapplication provides a method for the preparation of an immunogeniccomposition for the treatment and/or prophylaxis of mycoplasmainfections in a subject comprising a) cultivation of mycoplasma bacteriain a serum-reduced or swine serum free, eukaryotic cell system; b)obtaining an antigen of the mycoplasma bacteria; and c) addition of apharmaceutically acceptable carrier, wherein the eukaryotic cell systemcomprises a MDCK cell line. Again, the mycoplasma antigen can be wholeinactivated mycoplasma bacterin, preferably inactivated with formalin.

In a further aspect of the present invention the eukaryotic cell systemcomprises a McCoy cell line. The McCoy cell line is known to the personskilled in the art. Thus, according to one aspect, the presentapplication provides a method for the preparation of an immunogeniccomposition for the treatment and/or prophylaxis of mycoplasmainfections in a subject comprising a) cultivation of mycoplasma bacteriain a serum-reduced or swine serum free, eukaryotic cell system; b)obtaining an antigen of the mycoplasma bacteria; and c) addition of apharmaceutically acceptable carrier, wherein the eukaryotic cell systemcomprises a McCoy cell line. Again, the mycoplasma antigen can be wholeinactivated mycoplasma bacterin, preferably inactivated with formalin.

In one aspect of the present invention the mycoplasma bacteria areselected from the group consisting of: M. hyopneumoniae, M. hyorhinis,M. hyosynoviae and M. bovis. Thus, according to one aspect, the presentapplication provides a method for the preparation of an immunogeniccomposition for the treatment and/or prophylaxis of mycoplasmainfections in a subject comprising a) cultivation of mycoplasma bacteriain a serum-reduced or swine serum free, eukaryotic cell system; b)obtaining an antigen of the mycoplasma bacteria; and c) addition of apharmaceutically acceptable carrier, wherein the mycoplasma bacteria isselected from the group consisting of: M. hyopneumoniae, M. hyorhinis,M. hyosynoviae and M. bovis. Preferably the mycoplasma antigen can bewhole inactivated mycoplasma bacterin, preferably inactivated withformalin. It is understood, that only one, some or all of the mycoplasmaantigens can be whole inactivated bacterin.

In a further aspect of the present invention the mycoplasma bacteria areselected from the group consisting of: M. hyopneumoniae, M. hyorhinis,M. hyosynoviae and any combinations thereof. Thus, according to oneaspect, the present application provides a method for the preparation ofan immunogenic composition for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation ofmycoplasma bacteria in a serum-reduced or swine serum free, eukaryoticcell system; b) obtaining an antigen of the mycoplasma bacteria; and c)addition of a pharmaceutically acceptable carrier, wherein themycoplasma bacteria are selected from the group consisting of: M.hyopneumoniae, M. hyorhinis, M. hyosynoviae and any combinationsthereof. Preferably the mycoplasma antigen can be whole inactivatedmycoplasma bacterin, preferably inactivated with formalin. It isunderstood, that only one, some or all of the mycoplasma antigens can bewhole inactivated bacterin.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of M. hyopneumoniae bacteria in a serum-reduced or swineserum free, eukaryotic cell system; b) obtaining an antigen of the M.hyopneumoniae bacteria; and c) addition of a pharmaceutically acceptablecarrier. Preferably the antigen of M. hyopneumoniae is whole inactivatedM. hyopneumoniae bacterin, preferably inactivated with formalin. It isunderstood, that only one, some or all of the M. hyopneumoniae antigenscan be whole inactivated M. hyopneumoniae bacterin.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of M. hyorhinis bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the M.hyorhinis bacteria; and c) addition of a pharmaceutically acceptablecarrier. Preferably the antigen of M. hyorhinis is whole inactivated M.hyorhinis bacterin, preferably inactivated with formalin. It isunderstood, that only one, some or all of the M. hyorhinis antigens canbe whole inactivated M. hyorhinis bacterin.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of M. hyosynoviae bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the M.hyosynoviae bacteria; and c) addition of a pharmaceutically acceptablecarrier. Preferably the antigen of M. hyosynoviae is whole inactivatedM. hyosynoviae bacterin, preferably inactivated with formalin. It isunderstood, that only one, some or all of the M. hyosynoviae antigenscan be whole inactivated M. hyosynoviae bacterin.

According to a further aspect, the immunogenic composition comprisesmycoplasma antigen selected from the group consisting of: M.hyopneumoniae, M. hyorhinis, M. hyosynoviae and any combinationsthereof. At least bacteria of one of the mycoplasma species as describedabove (e.g. M. hyopneumoniae or M. hyorhinis or M. hyosynoviae) whichare used for the preparation of the mycoplasma immunogenic compositionare cultivated according to the invention in a serum-reduced or swineserum free, eukaryotic cell system. Preferably, all of the mycoplasmabacteria which are used for the preparation of the mycoplasmaimmunogenic composition are cultivated according to the invention in aserum-reduced or swine serum free, eukaryotic cell system.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition which comprisesmycoplasma antigen of M. hyopneumoniae, M. hyorhinis, M. hyosynoviaeand/or any combinations thereof for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation of at leastone mycoplasma bacteria selected from the group consisting of: M.hyopneumoniae, M. hyorhinis, and M. hyosynoviae in a serum-reduced orswine serum free, eukaryotic cell system; b) obtaining an antigen of themycoplasma bacteria; and c) addition of a pharmaceutically acceptablecarrier. Preferably all of the mycoplasma bacteria are cultivated in aserum-reduced or swine serum free, eukaryotic cell system according tothe invention. Preferably, at least one of the mycoplasma antigens iswhole inactivated mycoplasma bacterin, preferably inactivated withformalin. It is understood, that only one, some or all of the mycoplasmaantigens can be whole inactivated bacterin.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition which comprisesmycoplasma antigen of M. hyopneumoniae, M. hyorhinis, M. hyosynoviaeand/or any combinations thereof for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation of at leastM. hyopneumoniae bacteria in a serum-reduced or swine serum free,eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier.Preferably all of the M. hyopneumoniae bacteria are cultivated in aserum-reduced or swine serum free, eukaryotic cell system according tothe invention. Preferably, at least one of the M. hyopneumoniae antigensis whole inactivated M. hyopneumoniae bacterin, preferably inactivatedwith formalin. It is understood, that only one, some or all of the M.hyopneumoniae antigens can be whole inactivated bacterin.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition which comprisesmycoplasma antigen of M. hyopneumoniae, M. hyorhinis, M. hyosynoviaeand/or any combinations thereof for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation of at leastM. hyorhinis bacteria in a serum-reduced or swine serum free, eukaryoticcell system; b) obtaining an antigen of the mycoplasma bacteria; and c)addition of a pharmaceutically acceptable carrier. Preferably all of theM. hyorhinis bacteria are cultivated in a serum-reduced or swine serumfree, eukaryotic cell system according to the invention. Preferably, atleast one of the M. hyorhinis antigens is whole inactivated M. hyorhinisbacterin, preferably inactivated with formalin. It is understood, thatonly one, some or all of the M. hyorhinis antigens can be wholeinactivated bacterin.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition which comprisesmycoplasma antigen of M. hyopneumoniae, M. hyorhinis, M. hyosynoviaeand/or any combinations thereof for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation of at leastM. hyosynoviae bacteria in a serum-reduced or swine serum free,eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier.Preferably all of the M. hyosynoviae bacteria are cultivated in aserum-reduced or swine serum free, eukaryotic cell system according tothe invention. Preferably, at least one of the M. hyosynoviae antigensis whole inactivated M. hyosynoviae bacterin, preferably inactivatedwith formalin. It is understood, that only one, some or all of the M.hyosynoviae antigens can be whole inactivated bacterin.

In one aspect of the present invention the immunogenic composition isformulated for a single-dose administration. Thus, according to oneaspect, the present application provides a method for the preparation ofan immunogenic composition for the treatment and/or prophylaxis ofmycoplasma infections in a subject comprising a) cultivation ofmycoplasma bacteria in a serum-reduced or swine serum free, eukaryoticcell system; b) obtaining an antigen of the mycoplasma bacteria; and c)addition of a pharmaceutically acceptable carrier, wherein theimmunogenic composition is formulated for a single-dose administration.Preferably, at least one of the mycoplasma antigens is whole inactivatedmycoplasma bacterin, preferably inactivated with formalin. It isunderstood, that only one, some or all of the mycoplasma antigens can bewhole inactivated bacterin.

Advantageously, the experimental data provided by the present inventiondisclose that a single dose administration of the immunogeniccomposition of the present invention reliably and effectively stimulateda protective immune response. Specifically, a measurable antibodyresponse has been shown for M. hyorhinis and M. hyopneumoniae.

The term “subject” as used herein relates to animals, preferably tomammals such as mice, rats, guinea pigs, rabbits, hamsters, swine,sheep, dogs, cats, horses, monkeys, or cattle and, also preferably, tohumans.

In one aspect of the present invention the subject is a swine. Thus,according to one aspect, the present application provides a method forthe preparation of an immunogenic composition for the treatment and/orprophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the subject is a swine. Preferably, at least one of themycoplasma antigens is whole inactivated mycoplasma bacterin, preferablyinactivated with formalin. It is understood, that only one, some or allof the mycoplasma antigens can be whole inactivated bacterin.

In a further aspect of the present invention the subject is cattle.Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the subject is a cattle. Preferably, at least one of themycoplasma antigens is whole inactivated mycoplasma bacterin, preferablyinactivated with formalin. It is understood, that only one, some or allof the mycoplasma antigens can be whole inactivated bacterin.

In a further aspect of the present invention the subject is a cat. Thus,according to one aspect, the present application provides a method forthe preparation of an immunogenic composition for the treatment and/orprophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the subject is a cat. Preferably, at least one of the mycoplasmaantigens is whole inactivated mycoplasma bacterin, preferablyinactivated with formalin. It is understood, that only one, some or allof the mycoplasma antigens can be whole inactivated bacterin.

In a further aspect of the present invention the subject is a dog. Thus,according to one aspect, the present application provides a method forthe preparation of an immunogenic composition for the treatment and/orprophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the subject is a dog. Preferably, at least one of the mycoplasmaantigens is whole inactivated mycoplasma bacterin, preferablyinactivated with formalin. It is understood, that only one, some or allof the mycoplasma antigens can be whole inactivated bacterin.

The term “pharmaceutical-acceptable carrier” includes any and allsolvents, dispersion media, coatings, stabilizing agents, diluents,preservatives, antibacterial and antifungal agents, isotonic agents,adsorption delaying agents, adjuvants, immune stimulants, andcombinations thereof.

In one aspect of the present invention the pharmaceutical-acceptablecarrier is an adjuvant.

“Adjuvants” as used herein, can include aluminum hydroxide and aluminumphosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc.,Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham,Ala.), water-in-oil emulsion, oil-in-water emulsion,water-in-oil-in-water emulsion. The emulsion can be based in particularon light liquid paraffin oil (European Pharmacopea type); isoprenoid oilsuch as squalane or squalene; oil resulting from the oligomerization ofalkenes, in particular of isobutene or decene; esters of acids or ofalcohols containing a linear alkyl group, more particularly plant oils,ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryltri-(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, in particular isostearic acid esters. The oilis used in combination with emulsifiers to form the emulsion. Theemulsifiers are preferably nonionic surfactants, in particular esters ofsorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, ofpolyglycerol, of propylene glycol and of oleic, isostearic, ricinoleicor hydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, in particular thePluronic products, especially L121. See Hunter et al., The Theory andPractical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). JohnWiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570(1997). For example, it is possible to use the SPT emulsion described onpage 147 of “Vaccine Design, The Subunit and Adjuvant Approach” editedby M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59described on page 183 of this same book. Further suitable adjuvantsinclude, but are not limited to, the RIBI adjuvant system (Ribi Inc.),Block co-polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, EmeryvilleCalif.), monophosphoryl lipid A, Avridine lipid-amine adjuvant,heat-labile enterotoxin from E. coli (recombinant or otherwise), choleratoxin, IMS 1314 or muramyl dipeptide among many others. Among thecopolymers of maleic anhydride and alkenyl derivative, the copolymersEMA (Monsanto), which are copolymers of maleic anhydride and ethylene,are included. The dissolution of these polymers in water leads to anacid solution that will be neutralized, preferably to physiological pH,in order to give the adjuvant solution into which the immunogenic,immunological or vaccine composition itself will be incorporated.

A further example of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked, especially withpolyalkenyl ethers of sugars or polyalcohols. These compounds are knownby the term carbomer (Pharmeuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462 whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, preferably not more than 8,the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms, e.g.vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name CARBOPOL®; (BF Goodrich, Ohio,USA) are particularly appropriate. They are polymers of acrylic acidcross-linked with polyalkenyl ethers or divinyl glycol or cross-linkedwith an allyl sucrose or with allyl pentaerythritol. Among them, theremay be mentioned CARBOPOL® 974P, 934P and 971P. Most preferred is theuse of CARBOPOL® 971P.

Preferably, the adjuvant is added in an amount of about 100 μg to about10 mg per dose. Even more preferably the adjuvant is added in an amountof about 100 μg to about 10 mg per dose. Still more preferably theadjuvant is added in an amount of about 500 μg to about 5 mg per dose.Still more preferably the adjuvant is added in an amount of about 750 μgto about 2.5 mg per dose. Most preferably the adjuvant is added in anamount of about 1 mg per dose.

In a preferred embodiment of the invention the adjuvant is selected fromthe group consisting of aluminum hydroxide, aluminum phosphate,saponins, water-in-oil emulsion, oil-in-water emulsion,water-in-oil-in-water emulsion, polymers of acrylic or methacrylic acid,copolymers of maleic anhydride and alkenyl derivative, the RIBI adjuvantsystem, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridinelipid-amine, heat-labile enterotoxin from E. coli (recombinant orotherwise), cholera toxin, IMS 1314, muramyl dipeptide, and combinationsthereof.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the pharmaceutical-acceptable carrier is an adjuvant.Preferably, at least one of the mycoplasma antigens is whole inactivatedmycoplasma bacterin, preferably inactivated with formalin. It isunderstood, that only one, some, or all of the mycoplasma antigens canbe whole inactivated bacterin.

In one aspect of the present invention the pharmaceutically acceptablecarrier is a water-in-oil-in-water emulsion or a carbomer. Thus,according to one aspect, the present application provides a method forthe preparation of an immunogenic composition for the treatment and/orprophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the pharmaceutically acceptable carrier is awater-in-oil-in-water emulsion or a carbomer. Preferably, at least oneof the mycoplasma antigens is whole inactivated mycoplasma bacterin,preferably inactivated with formalin. It is understood, that only one,some, or all of the mycoplasma antigens can be whole inactivatedbacterin.

In one aspect of the present invention the water-in-oil-in-wateremulsion is Montanide ISA207 VG. Montanide ISA207 VG is an adjuvantcomposed of oleic esters of anhydrous mannitol in solution in a nonmineral oil and is designed to achieve water-in-oil-in-water vaccineemulsions. Montanide ISA207 VG is well known to the person skilled inthe art and can be used.

Thus, according to one aspect, the present application provides a methodfor the preparation of an immunogenic composition for the treatmentand/or prophylaxis of mycoplasma infections in a subject comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the pharmaceutically acceptable carrier is Montanide ISA207 VGor CARBOPOL®. Preferably, at least one of the mycoplasma antigens iswhole inactivated mycoplasma bacterin, preferably inactivated withformalin. It is understood, that only one, some, or all of themycoplasma antigens can be whole inactivated bacterin.

The present invention also relates to a method as described above forincreasing the immunogenicity of a mycoplasma antigen. Advantageously,the experimental data provided by the present invention disclose thatmycoplasma bacteria antigens provided by the above described method havean increased immunogenicity compared to antigens obtained frommycoplasma bacteria cultivated in a cell free culturing system.Specifically, MDCK-based M. hyorhinis vaccines showed earlier on-set ofsero-conversion, greater number of sero-positive pigs and higherserological titers.

Thus, according to one aspect, the present application provides a methodfor increasing the immunogenicity of an antigen comprising a)cultivation of mycoplasma bacteria in a serum-reduced or swine serumfree, eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier.Preferably, at least one of the mycoplasma antigens is whole inactivatedmycoplasma bacterin, preferably inactivated with formalin. It isunderstood, that only one, some, or all of the mycoplasma antigens canbe whole inactivated bacterin.

In one aspect of the present invention the antigen has an increasedimmunogenicity compared to an antigen obtained from mycoplasma bacteriacultivated in a cell free culturing system. Thus, according to oneaspect, the present application provides a method for increasing theimmunogenicity of an antigen comprising a) cultivation of mycoplasmabacteria in a serum-reduced or swine serum free, eukaryotic cell system;b) obtaining an antigen of the mycoplasma bacteria; and c) addition of apharmaceutically acceptable carrier, wherein the immunogenic compositionhas an increased immunogenicity compared to an immunogenic compositioncomprising the same antigen obtained from mycoplasma bacteria cultivatedin a cell free culturing system. Preferably, at least one of themycoplasma antigens is whole inactivated mycoplasma bacterin, preferablyinactivated with formalin. It is understood, that only one, some, or allof the mycoplasma antigens can be whole inactivated bacterin.

The term “increased immunogenicity” as used herein, means that theimmunological response caused by an immunogenic composition comprisingan antigen of interest is increased as compared to a referenceimmunogenic composition comprising the same antigen, wherein the antigenof the reference immunogenic composition is prepared of mycoplasmabacteria cultivated in a cell free culturing system.

The term “increased” means, that the cellular and/or antibody mediatedimmune response is increased by at least 10%, preferably by at least20%, more preferably by at least 30%, even more preferably by at least40%, even more preferably by at least 50%, even more preferably by atleast 75%, most preferably by at least 100% as compared to the cellularand/or antibody mediated immune response elicited by a referenceimmunogenic composition comprising the same antigen, wherein the antigenof the reference immunogenic composition is prepared of mycoplasmabacteria cultivated in a cell free culturing system. It is in thegeneral knowledge of a person skilled in the art how to measure thecellular and/or antibody mediated immune response. In particular, it isclear to such person skilled in the art either to compare the cellularmediated immune response of the immunogenic composition of interest withcellular mediated immune response of the reference, or the antibodymediated immune response of the immunogenic composition of interest withthat of the reference composition, but neither the cellular mediatedimmune response of a immunogenic composition of interest with theantibody mediated immune response of the reference or vice versa.Moreover, the cellular mediated immune response can be measured, forexample, by measuring the activation of cytotoxic T-cells by animmunogenic composition/antigen of interest. The antibody mediatedimmune response can be measured, for example, by measuring the amount ofantigen specific antibodies, generated in cause of the administration ofthe immunogenic composition comprising such antigen to an animal. Thecellular and/or antibody mediated immune response can be measured, forexample, by using a mouse model, a cat model, a cattle model or a swinemodel. However, the assays as described in Example 4 and 5 shall be usedas a reference assay for detecting the immunological response against M.hyorhinis and M. hyopneumoniae.

The term “same antigen” means, that nature of the antigens is identical.Thus, if the mycoplasma antigen of the immunogenic composition producedin a serum-reduced, eukaryotic cell system is whole inactivated bacterinof M. hyorhinis, than the same antigen means that the mycoplasma antigenof the cell-free system is also whole inactivated bacterin of M.hyorhinis. Furthermore, if the mycoplasma antigen of the immunogeniccomposition produced in a serum-reduced, eukaryotic cell system isprepared or purified according to a specific method, than the “sameantigen” means that the mycoplasma antigen of the cell-free system isprepared or purified according to the same method.

The term “reference” immunogenic composition refers to an immunogeniccomposition not obtained by the cultivation of mycoplasma bacteria in aserum-reduced or swine serum-free, eukaryotic cell system according tothe present invention.

Rather the term “reference” immunogenic composition refers to animmunogenic composition obtained by the cultivation of mycoplasmabacteria in a cell free culturing system supplemented with serum usingthe same antigen. The cultivation of mycoplasma bacteria in a cell freeculturing system supplemented with serum is well known to the personskilled in the art. The serum origin and the serum concentration aredependent on the mycoplasma bacteria to be cultivated and the yields ofmycoplasma bacteria to be obtained. M. hyorhinis, M. hyopneumoniae andM. hyosynoviae are generally cultivated in a cell free culturing systemsupplemented with 10-20% (v/v) porcine serum and 5-10% (v/v) yeastextract to obtain high yields. However, it is to be understood that theserum concentrations can be varied, the yeast extract can be deleted andthe origin of the serum can be changed, exemplary to fetal calf serum orthe like.

The present invention does not only provide methods for the preparationof immunogenic compositions or methods for increasing the immunogenicityof an antigen as defined above, it also relates to an immunogeniccomposition that is obtainable by the methods as described above. Thus,in a further aspect the present application relates to an immunogeniccomposition obtainable by a method according to the invention anddescribed herein. In general, such method comprises a) cultivation ofmycoplasma bacteria in a serum-reduced or swine serum free, eukaryoticcell system; b) obtaining an antigen of the mycoplasma bacteria; and c)addition of a pharmaceutically acceptable carrier. Preferably, at leastone of the mycoplasma antigens is whole inactivated mycoplasma bacterin,preferably inactivated with formalin. It is understood, that only one,some, or all of the mycoplasma antigens can be whole inactivatedbacterin.

In one aspect of the present invention the immunogenic composition showsan increased immunogenicity compared to a reference immunogeniccomposition comprising an antigen, wherein the antigen of the referenceimmunogenic composition is prepared of a mycoplasma bacteria cultivatedin a cell free culturing system.

Thus, according to a further aspect, the present application provides animmunogenic composition obtainable by a method comprising a) cultivationof mycoplasma bacteria in a serum-reduced or swine serum free,eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier.According to a further aspect, such immunogenic composition shows anincreased immunogenicity compared to a reference immunogenic compositioncomprising the same antigen prepared of a mycoplasma bacteria cultivatedin a cell free culturing system. Preferably, at least one of themycoplasma antigens is whole inactivated mycoplasma bacterin, preferablyinactivated with formalin. It is understood, that only one, some, or allof the mycoplasma antigens can be whole inactivated bacterin.

In a further aspect, the immunogenic composition comprises components ofthe eukaryotic cells of said eukaryotic cell system.

The term “components of the eukaryotic cells” comprises both whole cellsand fragments of said eukaryotic cells. The term “fragment” comprisesany parts of the eukaryotic cell such as parts of the cell membrane orintracellular organelles as a whole or parts thereof. However, the termfragment also encompasses any part of said eukaryotic cell comprisinglipids, proteins, sugars, DNA, RNA and the alike as well as combinationsthereof. Further, the components of the eukaryotic cells and themycoplasma antigen can either be in the immunogenic compositionseparately or attached to each other or a combination thereof.

Thus, according to one aspect, the present application provides animmunogenic composition obtainable by a method comprising a) cultivationof mycoplasma bacteria in a serum-reduced or swine serum free,eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein the immunogenic composition comprises components of theeukaryotic cells of said eukaryotic cell system. Preferably, at leastone of the mycoplasma antigens is whole inactivated mycoplasma bacterin,preferably inactivated with formalin. It is understood, that only one,some, or all of the mycoplasma antigens can be whole inactivatedbacterin.

In a further aspect, said components of the eukaryotic cells areattached to the mycoplasma antigen.

The term “attached” refers to any interaction, association, binding,adhering or linking of said components of the eukaryotic cells to themycoplasma antigen. Thus, the term attached encompasses any interactionsincluding indirect or direct, non-reversible or reversible, physical andchemical, electrostatic, and/or covalent bonds. Thus, it has to beunderstood that the components of the eukaryotic cells, for example, canbe bound to the mycoplasma antigen. However, it has to be understoodthat the components of the eukaryotic cells can also be linked to themycoplasma antigen(s). Such linking can be produced by several methodswell known to the person skilled in the art such as formaldehydetreatment and the like.

Thus, according to one aspect, the present application provides animmunogenic composition obtainable by a method comprising a) cultivationof mycoplasma bacteria in a serum-reduced or swine serum free,eukaryotic cell system; b) obtaining an antigen of the mycoplasmabacteria; and c) addition of a pharmaceutically acceptable carrier,wherein said components of the eukaryotic cells are attached to themycoplasma antigen. Preferably, at least one of the mycoplasma antigensis whole inactivated mycoplasma bacterin, preferably inactivated withformalin. It is understood, that only one, some or all of the mycoplasmaantigens can be whole inactivated bacterin.

Typically, when a bacterial antigen such mycoplasma bacterin is used theimmunogenic composition contains an amount of about 10³ to about 10¹⁰colony forming units (CFU) of the bacterial antigen per dose,preferably, about 10⁴ to about 10⁹ CFU of the bacterial antigen perdose, more preferably about 10⁵ to about 10⁶ CFU of the bacterialantigen per dose. If inactivated bacterin is used in the immunogeniccomposition, the CFU values refer to the amount of mycoplasma bacteriaprior to inactivation.

For example, the immunogenic composition of the present inventioncomprising antigens of M. hyopneumoniae are preferably used in amountsof about 10² to about 10¹⁰ CFU per dose, preferably about 10³ to about10⁹ CFU per dose, even more preferably in an amount of about 10⁴ toabout 10⁸ CFU per dose, most preferably in an amount of about 10⁵ toabout 10⁷ CFU per dose. The immunogenic composition of the presentinvention comprising antigens of M. hyorhinis are preferably used inamounts of about 10² to about 10¹⁰ CFU per dose, preferably about 10³ toabout 10⁹ CFU per dose, even more preferably in an amount of about 10⁴to about 10⁸ CFU per dose, most preferably in an amount of about 10⁵ toabout 10⁷ CFU per dose. The immunogenic composition of the presentinvention comprising antigens of M. hyosynoviae are preferably used inamounts of about 10² to about 10¹⁰ CFU per dose, preferably about 10³ toabout 10⁹ CFU per dose, even more preferably in an amount of about 10⁴to about 10⁸ CFU per dose, most preferably in an amount of about 10⁵ toabout 10⁷ CFU per dose.

Thus, according to one aspect, the present application provides methodfor providing an immunogenic composition comprising a) cultivation ofmycoplasma bacteria in a serum-reduced or swine serum free, eukaryoticcell system; b) obtaining an antigen of the mycoplasma bacteria; and c)addition of a pharmaceutically acceptable carrier, wherein theimmunogenic composition contains an amount of about 10³ to about 10¹⁰colony forming units (CFU) of the bacterial antigen per dose.Preferably, at least one of the mycoplasma antigens is whole inactivatedmycoplasma bacterin, preferably inactivated with formalin. It isunderstood, that only one, some or all of the mycoplasma antigens can bewhole inactivated bacterin.

It is understood by a person skilled in the art, that the variousprocess steps of the methods for providing an immunogenic composition asdescribed herein can be combined to practice the invention as describedherein.

EXAMPLES

The following examples are only intended to illustrate the presentinvention. They shall not limit the scope of the claims in any way.

Example 1 Cultivation of Mycoplasma Bacteria in MDCK Cells or McCoyCell, Respectively

A. Cultivation of M. hyorhinis, M. hyosynoviae and M. hyopneumoniae inMDCK Cells

M. hyorhinis:

Confluent T75 flask(s) of MDCK cells are trypsinized and subculturedinto 5, T150 flasks (1:10 split) using MEM+5% FBS. Flasks are incubatedat 37° C.+5% CO₂ until an approximately 95-100% confluent monolayer isobserved. Media is decanted and flasks rinsed twice with 1×PBS.Four-five mls of M. hyorhinis is added to each flask (MOI=10-100).Confluent cells in flasks are infected under the same incubationconditions as above for no less than 2 hours. After the infectionperiod, sufficient Infection Media (MEM+2% FBS), pre-warmed toapproximately 37° C., is added to each flask for a total volume of 60 mlper flask. Flasks are incubated until >90% CPE (approximately 3-7 days).Cell suspensions are collected from each flask and pooled together(Passage=n). The pooled material is used to infect new flasks ofapproximately 95-100% confluent MDCK cells in the same manner as theprevious infection (Passage=n+1), increasing the number of flasks usedto achieve a sufficient final volume as deemed necessary (Passage=n+2,Passage=n+3, etc).

M. hyosynoviae:

M. hyosynoviae is cultured in the same manner as M. hyorhinis with a fewmodifications: Infection Media contains DMEM+2% FBS+1% argininesolution; M. hyosynoviae does not typically exhibit CPE so color changeand turbidity of the media is the key indicator to subculture to thenext passage.

M. hyopneumoniae:

M. hyopneumoniae is cultured in the same manner as M. hyorhinis.Depending on the strain used for infection, CPE may or may not bepresent. Therefore, color change and turbidity of media can be used asthe indicator to subculture to the next passage.

B. Cultivation Conditions for M. hyorhinis, M. hyosynoviae and M.hyopneumoniae in McCoy Cells.

M. hyorhinis:

McCoy cells are grown as suspension cultures in stir flasks in modifiedEMEM supplemented with 10% FBS. Cells are subcultured by seeding newflasks so as to have a final concentration of 10⁵-10⁶ cells/ml. For M.hyorhinis, a 500 ml cell suspension at a concentration of 10⁵-10⁶cells/ml in a 3 L flask is seeded with 1 ml 10⁷-10⁸ CFUs. Flasks areincubated at 37° C. in the presence of 5% CO₂ on a magnetic stir platefor 3-7 days. Mycoplasma growth is ascertained by visible acidic pHchange and increase in turbidity. Mycoplasma growth is also evaluated byPFU assays to determine counts.

M. hyosynoviae:

M. hyosynoviae is cultivated in a similar manner as M. hyorhinis. A 500ml cell suspension at a concentration of 10⁵-10⁶ cells/ml in a 3 L flaskis seeded with 1 ml 10⁵-10⁷ CFUs. Flasks are then incubated at 37° C. inthe presence of 5% CO₂ on a magnetic stir plate for approximately 2weeks. Both pH change and increase in turbidity are used to determinegrowth in addition PFU assays to determine counts.

M. hyopneumoniae:

M. hyopneumoniae is cultivated in a similar manner as M. hyorhinis andM. hyosynoviae. A 500 ml cell suspension at a concentration of 10⁵-10⁶cells/ml in a 3 L flask is seeded with 1 ml 10⁵-10⁷ CFUs. Flasks areincubated at 37° C. in the presence of 5% CO₂ on a magnetic stir platefor approximately 2 weeks. Both pH change and increase in turbidity canbe used to determine growth in addition to PFU assays to determinecounts.

C. Cultivation of Mycoplasma Species with Various Serum Types

To evaluate whether a mycoplasma species can be cultivated in serum fromdifferent species, MDCK cells are infected with M. hyorhinis andcultured in either Fetal Bovine Serum, Porcine Serum, Rabbit Serum,Chicken Serum or Horse Serum. For each serum type, the cultivation of M.hyorhinis in MDCK cells is performed as described above (i.e., 5% serumfor cell growth and 2% serum for infection). M. hyorhinis is harvestedat four days post-infection per standard method. A CCU (color changingunit) assay is performed to determine the live titer of M. hyorhinis.Further, a qPCR (quantitative real time Poylmerase chain reaction) isperformed to determine the total genomic content of M. hyorhinis. Anexemplary experiment is shown in Table 1.

TABLE 1 Cultivation of M. hyorhinis with various serum types qPCR logCCU50 Serum type (gc/μl) (log/ml) Fetal Bovine Serum 6.15 8.00 PorcineSerum 6.06 8.50 Rabbit Serum 6.11 8.33 Chicken Serum 6.31 8.00 HorseSerum 6.49 9.00

Table 1 demonstrates that the titers measured either by CCU assay orqPCR are similar for the various serum types. Further, western blot data(not shown) support this data. Thus, M. hyorhinis can be cultivated inMDCK cells irrespective of which serum type is used for cultivation.

Example 2 Preparation of Vaccines

When final passage is ready to harvest (>90% CPE present), a singlefreeze-thaw cycle is performed on all flasks by placing them into a<−60° C. freezer for >2 hours, quickly thawing at 37° C., collecting andpooling lysate, and pipetting up and down several times to homogenize.Generally, 10-20% glycerol is then added to the suspension andhomogenized. The suspension is aliquoted into working volumes. Stocksare kept at <−60° C. until needed.

Appropriate volumes of the above stocks are inactivated with 0.2%formalin. Excess formalin is neutralized with sodium bisulfite at thetime of vaccine blending. Vaccines are blended with Montanide™ ISA 207VG adjuvant or with CARBOPOL® adjuvant. Vaccines are stored at 2-7° C.

Example 3 Assessment of Effectiveness of the Vaccines

The efficacy of vaccines is evaluated based on the ability to induce anantibody response (as well as the titer by ELISA) after administrationin swine.

Animal Care:

Animals are in good health and nutritional status before a study isinitiated. Prior to the randomization procedure a health examination isconducted. Non-medicated feed is used throughout the duration of thestudy. Feed rations are appropriate for the age, condition, and speciesof test animal according to facility standard operating procedure. Wateris provided ad libitum throughout the study.

Assessment of Effectiveness of M. hyorhinis and M. hyopneumoniaeVaccines After Administration in Swine.

M. hyorhinis:

On D0 and again on D21, conventional piglets of 6 weeks±5 days of ageare administered a 2 ml dose (7.1-7.3 log10 CCU/dose) of M. hyorhinisvaccine intramuscularly. M. hyorhinis is prepared as above; i.e.,cultured in MDCK cells as described above. The vaccine is adjuvantedwith Montanide ISA207VG or CARBOPOL®. PBS is used as Placebo. The pigsare observed daily for general health. Blood is collected prior tovaccination at D0, 7, 14, 21, 28, 35 and 42. The serum is tested for M.hyorhinis specific antibodies by BIVI R&D Indirect ELISA. For the BIVIR&D ELISA, an S/P ratio of >0.200 is considered positive.

In the example shown in Table 2, the M. hyorhinis ELISA indicates astrong antibody response. Six/six (100%) animals vaccinated with M.hyorhinis-MDCK+Montanide ISA207VG were positive two weeks after thefirst dose (D14). All animals remained positive through D42, with aboost in titers noted one week after the second dose (D28). Animalsvaccinated with M. hyorhinis-MDCK+CARBOPOL® also show an antibodyresponse.

TABLE 2 M. hyorhinis ELISA results Pre D0 D7 D14 D21 D28 D35 D42 M.hyorhinis 0.00 0.003 0.043 0.972 1.123 1.340 1.152 1.133 MDCK +Montanide ISA207VG M. hyorhinis 0.002 0.018 0.059 0.095 0.122 0.3540.375 0.409 MDCK + CARBOPOL ® Placebo (PBS) 0.001 0.005 0.011 0.0150.021 0.031 0.057 0.070

Similar antibody response results post-vaccination were achieved usingM. hyorhinis cultured in McCoy cells (data not shown). Similar resultswere obtained after a single dose administration (data not shown).

M. hyopneumoniae:

On D0 and again on D21 conventional piglets of 6 weeks±5 days of age areadministered a 2 ml dose (8.0-8.5 log10 CCU/dose) of M. hyopneumoniaevaccine intramuscularly. The vaccine is adjuvanted with MontanideISA207VG. PBS is used as Placebo. The pigs are observed daily forgeneral health. Blood is collected prior to vaccination at D0, 7, 14,21, 28, 35 and 42 to test for the presence of M. hyopneumoniaeantibodies. In one example, a commercial IDEXX ELISA was used. For theIDEXX ELISA, an S/P ratio of >0.400 was considered positive.

In the example shown in Table 3, the M. hyopneumoniae ELISA indicated astrong antibody response. For the M. hyopneumoniae MDCK+ISA207vaccinated animals, 3/6 (50%) animals were positive on D14 and 5/6(83.3%) on D21 with 6/6 (100%) positive on D28, 35, and 42.

TABLE 3 M. hyopneumoniae IDEXX ELISA results Pre D0 D7 D14 D21 D28 D35D42 M. hyopneumoniae −0.024 −0.022 0.034 0.601 0.949 1.775 1.986 1.895MDCK + Montanide ISA207VG Placebo (PBS) −0.019 0.011 −0.021 −0.015−0.025 −0.025 0.016 0.016

Similar results were obtained after a single dose administration, datanot shown.

Example 4 Effectiveness of Vaccines Obtained from Mycoplasma BacteriaCultured in a Eukaryotic Cell Line Versus Vaccines Obtained fromMycoplasma Bacteria Cultured in a Cell Free System

Fifty-four CD/CD animals at 8 weeks±5 days of age are divided into sixgroups. Group V1 and V2 each receive an inactivated MHRN001 isolate ofM. hyorhinis cultured in MDCK cells and CM (complex medium; such asproteose peptone based medium containing porcine serum and yeast extractor Friis-based media), respectively; Group V3 and V4 receive aninactivated MHRN002 isolate of M. hyorhinis cultured in MDCK cells andCM, respectively. All vaccines are adjuvanted with Montanide ISA207VG;dosage and route were 2×2 ml doses by intramuscular injection with onedose on D0 and the second on D21. Group CC (control group) receive anantigen-free placebo (PBS) in the same fashion. Group SC (strictcontrol) receive no treatment throughout the study, serving as strictcontrol animals. On D42, 43, and 44, pigs in Group V1-V4 and CC arechallenged with a virulent M. hyorhinis. Dosage and route ofadministration is 40 ml intraperitoneal, 15 ml intravenous, and 15 mlintranasal, respectively. Blood is collected weekly from D0 through theend of the study (D58) for M. hyorhinis-specific ELISA testing. For theR&D M. hyorhinis ELISA, an S/P ratio of >0.200 was considered positive.In the exemplary study shown in Table 4, all pigs in Group SC remainednegative throughout the study, indicating a lack of exposure to M.hyorhinis. Groups V I-V4 and CC were negative on D0 and 7. However,serology results varied between the CM-based and MDCK-based vaccines.When compared to the CM-based vaccines, the MDCK-based vaccines showedearlier on-set of sero-conversion, greater numbers of sero-positivepigs, and higher serological titers.

TABLE 4 M. hyorhinis ELISA results M. hyorhinis ELISA Average S/P Ratiosby Group Group DO D7 D14 D21 D28 D35 D42 D49 D58 MHRN001- −0.002 0.0100.199 0.482 0.916 0.971 0.929 1.056 0.991 MHRN001- 0.001 0.009 0.0310.081 0.395 0.401 0.370 0.793 0.770 MHRN002 0.004 0.014 0.157 0.4240.981 1.023 0.953 1.097 1.086 MDCK (V3) MHRN002- 0.003 0.011 0.017 0.0470.298 0.299 0.263 0.704 0.608 CM (V4) Placebo 0.004 0.004 0.009 0.0230.021 0.029 0.031 0.262 0.433 (CC) Strict −0.003 −0.002 −0.003 0.0050.008 0.015 0.021 0.031 0.060 Control (SC)

Titration experiments are performed to compare cell culture-basedvaccines to vaccines from mycoplasma bacteria cultured in a cell freesystem.

M. hyorhinis is cultured in McCoy cells as described above or culturedin CM (complex medium), respectively.

Vaccines are made with McCoy antigen using undiluted antigen (full),1:10 antigen, and 1:100 antigen, all blended 1:1 with Montanide ISA207VGas adjuvant. Vaccines using CM derived antigen are made in the samemanner.

Pigs (three weeks of age at the time of vaccination) are vaccinated witha single 2 mL dose administered IM on Day 0.

As shown in Table 5, group averages from the exemplary study were all“negative” prior to challenge (D0-D21), though, the “McCoy+ISA full” and“McCoy+ISA 1:10” showed responses trending towards positive. At one weekpost-challenge (D28), all vaccinate groups responded with the exceptionof CM 1:100.

From Table 5 it is apparent that each antigen type (McCoy, CM) exhibitsa standard titration effect (Full>1:10>1:100). Further, from Table 5 itis apparent that the “McCoy+ISA full” and “McCoy+ISA 1:10” vaccinateshave higher average scores than the “CM+ISA full” antigen, and thatholds true through termination at D42. Groups with positive (S/P>0.200)averages post-challenge are the “McCoy+ISA full” and the “McCoy+ISA1:10” groups. The McCoy Full vaccine and 1:10 dilution showed a highersero-response than CM Full vaccine both pre-challenge andpost-challenge. Furthermore, animals vaccinated with McCoy 1:100 alsodemonstrated a response distinguishable from placebo (non-vaccinates)and CM 1:100 groups (CM 1:100 response, or lack thereof, was equivalentto non-vaccinates). The titration experiments demonstrated that cellculture-based vaccines had better serological results compared tovaccines from mycoplasma bacteria cultured in a cell free system.

TABLE 5 M. hyorhinis ELISA results M. hyorhinis ELISA Average S/P Ratiosby Group Group DO D7 D14 D21 D28 D35 D42 McCoy + 0.002 0.012 0.032 0.1170.312 0.298 0.325 ISA full McCoy + 0.000 0.005 0.065 0.107 0.291 0.1930.221 ISA 1:10 McCoy + 0.001 0.001 −0.001 0.000 0.145 0.118 0.150 ISA1:100 CM + 0.002 0.009 0.011 0.034 0.190 0.163 0.190 ISA full CM +−0.001 0.017 0.003 0.014 0.114 0.132 0.176 ISA 1:10 CM + −0.002 0.000−0.002 0.001 0.019 0.043 0.093 ISA 1:100 Placebo: −0.001 0.002 −0.002−0.002 0.021 0.039 0.081 PBS + ISA Strict −0.002 −0.001 −0.002 −0.002−0.002 0.008 0.024 Control

The invention claimed is:
 1. A method for the preparation of animmunogenic composition for the treatment of mycoplasma infections in asubject comprising a bacterin comprising one or more antigens selectedfrom M. hyorhinis, M. hyosynoviae, and M. hyopneumoniae, and anycombination thereof, wherein at least one of bacteria M. hyorhinis, M.hyosynoviae, and/or M. hyopneumoniae is co-cultivated in a eukaryoticcell system comprising McCoy cells comprising the steps of: a.cultivating mycoplasma in a serum-reduced, McCoy cell system; b.obtaining such a mycoplasma bacteria as a bacterin comprising mycoplasmaantigens; and c. adding a pharmaceutically acceptable carrier.
 2. Themethod of claim 1, wherein the serum is free of swine serum.
 3. Themethod of claim 1, wherein the mycoplasma bacteria are cultivated in theabsence of serum.
 4. The method of claim 1, wherein the antigen has anincreased immunogenicity compared to an antigen obtained from mycoplasmabacteria cultivated in a cell free culturing system.
 5. The method ofclaim 1, wherein the bacterins are formalin or binary ethylenimine (BEI)inactivated bacterins.
 6. The method of claim 1, wherein the immunogeniccomposition comprises a bacterin comprising a combination of antigens ofM. hyorhinis and M. hyopneumoniae.
 7. The method of claim 1, wherein theimmunogenic composition comprises a bacterin comprising a combination ofantigens of M. hyopneumoniae, M. hyorhinis and M. hyosynoviae.
 8. Themethod of 1, wherein said immunogenic composition is formulated for asingle-dose administration.
 9. The method of claim 1, wherein saidsubject is a swine.
 10. The method of claim 1, wherein saidpharmaceutically acceptable carrier is selected from the groupconsisting of solvents, dispersion media, coatings, stabilizing agents,diluents, preservatives, antibacterial and antifungal agents, isotonicagents, adsorption delaying agents, adjuvants, immune stimulants, andcombinations thereof.
 11. The method of claim 1, wherein saidpharmaceutically acceptable carrier is an adjuvant selected from thegroup consisting of aluminum hydroxide, aluminum phosphate, saponins,water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-wateremulsion, polymers of acrylic or methacrylic acid, copolymers of maleicanhydride and alkenyl derivative, the RIBI adjuvant system, Blockco-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine,heat-labile enterotoxin from E. coli (recombinant or otherwise), choleratoxin, IMS 1314, muramyl dipeptide, and combinations thereof.
 12. Themethod of claim 1, wherein said pharmaceutically acceptable carrier is awater-in-oil-in-water emulsion or a carbomer.